In tomato (Lycopersicon esculentum Mill.), the coat protein (CP) gene of the white leaf strain of cucumber mosaic virus (CMV-WL) conferred a high level of resistance against American, Asian, European, and Oceanian strains belonging to both sero-groups of CMV. An analysis of genetic populations deriving from crosses and back-crosses of a homozygous CMV-resistant tomato line (TT5-007-11) with susceptible cultivars revealed that (1) the high level of resistance is conferred by a single dominant gene to which the symbol Cmv is assigned; (2) in grafts between CMV-resistant and -infected plants, the resistant plants developed systemic symptoms, indicating that they are not immune; (3) the CMV resistance is independent of the virus inoculum titer, and it can be effectively used for the production of F1 commercial hybrids; (4) the two markers, neomycin phosphotransferase II (NPT-II) and (β-glucuronidase (GUS), present in transgenic plants are not completely reliable for predicting resistance; and (5) Cmv confers resistance to most CMV strains containing satellltes (RNA5). but one mutant satellite derived from CMV-WL infected transgenic plants. This is the first report of a satellite that can interfere with the function of a CP gene. The valuable breeding line TT5-007-11 is resistant also to tobacco mosaic virus (Tm-22), Verticillium wilt, and Phytophthora infestans (Race 0

Gonsalves, D.

Provvidenti, R.

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SDU a TiranaLeaff S t a i dR. Provvidenti and D. Gonsalvesml® Tomato Lira£iiim Gdnnxg ©f th®In tomato (Lycopersicon esculentum Mill.), the coat protein (CP) gene of the whiteleaf strain of cucumber mosaic virus (CMV-WL) conferred a high level of resistanceagainst American, Asian, European, and Oceanian strains belonging to both sero-groups of CMV. An analysis of genetic populations deriving from crosses and back-crosses of a homozygous CMV-resistant tomato line (TT5-007-11) with susceptiblecultivars revealed that (1) the high level of resistance is conferred by a single dom-inant gene to which the symbol Cmv is assigned; (2) in grafts between CMV-resis-tant and -infected plants, the resistant plants developed systemic symptoms, in-dicating that they are not immune; (3) the CMV resistance is independent of thevirus inoculum titer, and it can be effectively used for the production of F1 com-mercial hybrids; (4) the two markers, neomycin phosphotransferase II (NPT-II) andp-glucuronidase (GUS), present in transgenic plants are not completely reliable forpredicting resistance; and (5) Cmv confers resistance to most CMV strains con-taining satellites (RNA5), but one mutant satellite derived from CMV-WL infectedtransgenic plants. This is the first report of a satellite that can interfere with thefunction of a CP gene. The valuable breeding line TT5-007-11 is resistant also totobacco mosaic virus (Tm-2>), Verticillium wilt, and Phytophthora infestans (Race 0).From the Department of Plant Pathology (Virology),Cornell University, New York State Agricultural Exper-iment Station, Geneva, NY 14456.Journal of Heredity 1995:86:85-88: 0022-1503/95/S5.00For several decades, all attempts to incor-porate resistance to cucumber mosaic virus(CMV) in tomato (Lycopersicon esculentumMill.) were without success. Althoughsources of resistance to this virus werefound in some wild tomatoes (L. chees-manii, L hirsutum, L. parviflorum, L pen-nellii, L peruvianum, and Solanum lycoper-sicoides), the polygenic nature ofresistance factors and incompatibility be-tween donors and receivers created greatdifficulties in exploiting them (Kariyamaet al. 1971; Latterot 1980; Phills et al. 1977;Watterson 1993).In 1973, a simple way to combine DNAfrom two different organisms and then cloneidentical copies gave birth to genetic en-gineering. Ten years later, a practical andefficient way for foreign genes to be intro-duced and expressed in plant species wasreported. This was accomplished by elim-inating (disarming) tumor-causing genesfrom the Ti plasmid of Agrobacterium tu-mefaciens and by inserting foreign genes.Hence, by leaving intact the DNA transfermechanism, it was possible to incorporateuseful genes into a variety of plant cells(Leemans 1993).For viral diseases, the most commonform of biotechnological control involvesthe insertion of the cDNA of a coat protein(CP) gene into a plant genome (Grumet1990). The exploitation of this "coat pro-tein mediated protection" or "transgeniccross-protection" has been attempted forseveral plant species (Beachy et al. 1990).For tomato, strains of CMV causing prom-inent symptoms were the major targets,but the resulting CP genes have proven tobe viral titer dependent, serogroup specif-ic, or only to condition a low level of re-sistance (Quemada et al. 1991). Conse-quently, in the past 2 years, our effortswere directed toward the utilization of aversatile CP gene that had derived fromCMV-WL, a member of subgroup II of theCucumovirus Group (Namba et al. 1991).Ordinarily, this strain incites a bright whit-ish mosaic on tomato leaves (WL = WhiteLeaf), but when its RNA5 satellite is re-moved by purification or through passagein cucurbits, the virus causes only mildsymptoms (Gonsalves et al. 1982). In to-bacco, the CMV-WL CP gene provided abroad spectrum of protection againststrains of both serotypes of the virus(Namba et al. 1991). Recently, this CP genewas incorporated into tomato and ap-peared to be as effective as a natural re-sistance gene (Xue et al. 1994). Thus, the85purpose of this study was to determinethe inheritance of resistance of the CMV-WL CP gene in tomato and assess the in-fluence of factors that may affect the ex-pression of resistance.Materials and MethodsThe transgenic tomato line R3 TT5-007-11,used in our study, is homozygous for re-sistance to CMV, tobacco mosaic virus(Tm-22}, Verticillium wilt, and Phytophthorainfestans, Race 0. Plants of this line alsocontained two useful markers, neomycinphosphotransferase II (NPT-II) and p-glu-curonidase (GUS) (Xue et al. 1994). For ge-netic studies, plants of this line werecrossed with Geneva 80 (G-80), which wasused for transformation (Xue et al. 1994),and Veemore (early maturity with mediumsize fruits). Plants of F,, F2, and reciprocalbackcross populations of these crosseswere grown in restricted areas in green-houses, and mechanically inoculated atthe two leaf stage with CMV-China, a mem-ber of subgroup 1 of the CucumovirusGroup (Kearney et al. 1990). Inoculum wasprepared by triturating CMV-infected G-80leaves with 0.05 M phosphate buffer (K+)at pH 8.5 and used at a dilution 1:10. Tominimize escapes, all plants were reino-culated on the third and fourth leaves. Todetermine the effectiveness of the CMV-WL CP gene, at least 16 plants of TT5-007-11 were tested with each of 13 strains ofthe virus originally collected in Australia(CMV-Y), California (CMV-CA), Egypt (CMV-E), Florida (CMV-FL), France (CMV-F), Ha-waii (CMV-H), Japan, (CMV-J), Mexico(CMV-M), New York (CMV-WL, CMV-LSS,and CMV-L2), New Zealand (CMV-NZ), andTaiwan (CMV-TW). Nine of these strainsbelonged to subgroup 1 (CMV-J, CMV-CA,CMV-E, CMV-F, CMV-FL, CMV-H, CMV-J,CMV-NZ, and CMV-TW) and four to sub-group II (CMV-WL, CMV-LSS, CMV-L2, andCMV-M) of Cucumovirus. Some of thesestrains were associated with satellites,thus offering the opportunity to test therole of specific RNA5 on the expression ofthe CP gene. Healthy plants of TT5-007-11were also grafted with CMV-infected G-80plants, to assess the type of resistance byintroducing in the healthy phloem a hightiter of the virus. To determine its suit-ability as a parent for the production ofcommercial F, hybrids, additional plantsof this line were crossed with those of thecv. Solarita (late maturity and large fruits).The polymerase chain reaction (PCR)technique was used to detect NPT-II, andthe GUS assays were performed with theTable 1. Inheritance of resistance to cucumbermosaic virus in the transgenic tomato lineTT5-007-11 possessing the coat protein gene ofCMV-WL strainParents andprogenyTT5-007-11 (007-11)Geneva 80 (G-80)VeemoreF, (G-80 x 007-11)F, (007-11 X G-80)F2 (007-11 x G-80)F, (007-11 x Veemore)F, (Veemore X 007-11)F2 (Veemore X 007-11)BC (Veemore X 007-11)X VeemoreBC (Veemore X 007-11)x 007-11ObservedsegregationRe-sis-tant56002735814537852485Sus-cep-tible0475800230035300Ex-pec-tedratio3:13:11:1Good-ness-of-fit(?).49.30.43fluorescent methyl umbelliferon product,which was visualized under an ultravioletlight. The CMV-WL CP gene was detectedby enzyme-linked immunosorbent assay(ELISA) using the homologous antiserum(Gonsalves et al. 1982; Xue et al. 1994). Allplants were kept in a restricted area of anaphid-free greenhouse, held at 25°C-28°C.ResultsInheritancePlants of TT5-007-11 inoculated with CMV-China reacted with a symptomless infec-tion restricted to the inoculated leaves.Recovery tests and ELISA involving all theother leaves and stem established that thevirus had failed to move systemically (sys-temic resistance). Plants of the suscepti-ble parents G-80, and Veemore, after an in-cubation period of 8-10 days, developedsystemic green mottle, leaf reduction, ne-crotic spotting, and stunting. From thedata presented in Table 1, it is evident thatall plants of F, TT5-007-11 x G-80, G-80 xTT5-007-11, TT5-007-11 x Veemore, Vee-more X TT5-007-11 failed to develop sys-temic infection, confirming that the resis-tance was dominant regardless of whichwas the maternal parent. Plants of the F2populations deriving from the abovecrosses segregated in the ratio 3 resistantto 1 susceptible. Further confirmation ofthe presence of a single dominant factorwas obtained from backcross populations.All plants of (TT5-007-11 x G-80) x TT5-007-11, and (TT5-007-11 x Veemore) XTT5-007-11 were systemically resistant.Conversely, plants of the testcross popu-lations (TT5-007-11 x G-80) x G-80, and(TT5-007-11 x Veemore) x Veemore seg-regated approximately in the ratio 1resistant to 1 susceptible. Consequently,to the CP gene conferring monogenicallydominant resistance to CMV in tomato,the symbol Cmv is assigned.Resistance to CMV Subgroups I and IIWhen groups of 16 plants of TT5-007-11were inoculated with each of the nine Cuc-umovirus strains belonging to subgroup I(CMV-CA, CMV-E, CMV-F, CMV-FL, CMV-H,CMV-J, CMV-NZ, CMV-TW, and CMV-Y) andthree to subgroup II (CMV-WL, CMV-L2,and CMV-M) all were found to be system-ically free of the virus. ELISA and recoverytests clearly showed that these virusesfailed to move systemically in stems andleaves of TT5-007-11. The resistance con-ferred by Cmv did not depend on the virusinoculum titer, since it was able to with-stand a minimum dilution of 1 (infectedplant sap) to 1 (buffer) with any of thestrains listed above.Type of Resistance Conferred by theCMV CP GeneRecovery tests and ELISA had confirmedthat the Cmv gene conferred a high levelof systemic resistance in TT5-007-11plants. However, when 12 of these resis-tant plants were individually approach-grafted to CMV-infected G-80 plants, with-in 12 days resistant plants developedsystemic mosaic and stunting. Identical re-sults were obtained when a dozen apicalportions (6-8 cm) of healthy TT5-007-11plants were individually splice-grafted onCMV-infected plants. Local lesion tests onChenopodium quinoa and ELISA revealed avery high titer of the virus in leaf and stemtissues of infected TT5-007-11 plants.Thus, these tests demonstrated that by in-troducing a steady flow of virus into thephloem, resistant transgenic plants suc-cumbed to infection. Thus, resistance wasnot at the immunity level.Effect of the RNA5 Satellites on CPGenePlants of TT5-OO7-11 were fully systemical-ly resistant to the following CMV strainspossessing specific RNA5-satellites: CMV-WL+RNA5-WL (white leaf), CMV-China+RNA5-N (necrotic leaf), and CMV-Mexi-co+RNA5-ML (mild leaf). However, a mu-tant recently derived from CMV-WL infect-ed plants of TT5-OO7-11, causing severestunting and drastic reduction of leaf lam-inae, thereby giving the appearance ofshoe-strings (LSS). When CMV-LSS andCMV-WL were individually cultured incucumber (Cucumis sativus L.) or summer86 The Journal of Heredity 1995:86(2)squash (Cucurbita pepo L.) the helper CMVreplicated normally, but the satellites(RNA5-WL and RNA5-LSS) replicated veryslowly and were easily eliminated througha series of transfers in these hosts (un-published data). Both CMV isolates lack-ing the satellite RNAs caused on suscep-tible plants a very mild chlorotic mottlingof foliage and no plant stunting. Converse-ly, plants of TT5-007-11 inoculated withthese CMV isolates free of satellites re-mained symptomless, and recovery testsand ELISA confirmed the absence of viralinfection in leaves and stems. Thus, it wasevident that by eliminating the RNA5-LSSsatellite, the helper CMV failed to infectplants possessing the Cmv gene.Marker Genes for Predicting CMVResistanceAlthough the two marker genes, NPT-IIand GUS, were useful during the develop-ment of CMV-resistant transgenic plants,they were not completely reliable for pre-dicting resistance. Among plants found tobe resistant to CMV in F2 populations, 596-15% of them were negative for one or bothof these markers. Also, in the same F2 pop-ulations, there was a similar percentage ofplants which were positive for one or bothof these markers, but were susceptible toCMV. Although resistance to the virus fol-lowed Mendelian segregation, the markersdid not. Thus, the CMV inoculationsproved to be more reliable in indicatingwhich plants in segregating populationspossessed the Cmv gene.Resistant Line as a Parent of F, HybridsPlants of TT5-007-11 were crossed with So-larita, a cultivar of late maturity whichproduces large fruits and has Verticilliumand Fusarium wilt resistance. The resulting(TT5-007-11 x Solarita)F, or (Solarita XTT5-007-ll)F, plants produced large fruits;were resistant to CMV, TMV, Fusarium wilt,Verticillium wilt, and late blight; and wereof intermediate maturity.DiscussionFor the first time, it was possible, throughgenetic engineering, to incorporate in to-mato a gene conferring a high level of re-sistance to CMV (Xue et al. 1994). Thissingle dominant gene is comparable to anatural resistance gene; thus, it can beeasily incorporated in other cultivars us-ing backcrossing or pedigree breeding. Ananalysis of transgenic resistant tomatoplants revealed: (1) The Cmv gene offersvery effective protection against a numberof CMV strains from America, Asia, Eu-rope, and Oceania, which belong to thetwo known serogroups of the virus. Thisresistance is independent of the virus in-oculum titer, since it can withstand a min-imum dilution of 1 (infected plant sap) to1 (buffer) with any strain of this virus. (2)Preliminary tests have already shown thatthe homozygous line TT5-OO7-11 is wellsuited for the production of F, hybrids, be-cause it is homozygous resistant to CMV,TMV, Verticillium wilt, and late blight(Race 0). (3) The two marker genes, NPT-IIand GUS, associated with the Cmv geneare still useful, but they are not complete-ly reliable; hence, during breeding, thescreening for resistance must be basedupon viral testing. (4) Recovery tests andELISA confirmed that the Cmv gene con-fers a high level of resistance, but graftsbetween resistant and CMV-infected plantsdetermined that this resistance is not im-munity. (5) Although Cmv conferred resis-tance to strains of CMV possessing threedistinct satellites (RNA5-WL, RNA5-N, andRNA5-ML), a mutant (RNA5-LSS), derivingfrom RNA5-WL, infected transgenic resis-tant plants.There is already evidence that a CP genecan be effective in one plant species andpartially or totally ineffective in others.Our CMV-WL CP gene appeared to be farless effective when it was introduced intomelons (Cucumis melo L.) (Gonsalves et al.1994). Even in tomato, the best resultswere obtained with our early ripeningTMV-resistant cultivar Geneva 80, whichwas developed several years ago (Xue etal. 1994). This suggests that, in this culti-var, the gene found a stable chromosomallocus, which allows a full expression of itsfunction.This study has given the first indicationthat a satellite can interfere with the func-tion of a CP gene. It was evident that Cmvgene was able to control the CMV sharedby two strains, but of the two associatedsatellites (RNA5-WL and RNA5-LSS), onlyone (RNA5-LSS) interfered with the func-tion of the resistance gene. It is alreadyknown that in satellites minor nucleotidesequence changes can be responsible foraltering host responses (Palukaitis 1988).The performance of transgenic cropssuch as the one described in this articlehas induced private, as well as state insti-tutions to seek deregulation from theUSDA, thus treating them as conventionalcrops. Environmentalists, however, arenot entirely convinced that the ecologicalrisks are minimal. One of their major fearsis that these "man-assembled" genes willescape from transgenic crops via pollina-tion and become incorporated into wildspecies. They remind us that in many ar-eas of the world, exotic plant species havebecome serious pests. In a similar manner,engineered plants may become the sourcesof exotic traits causing adverse economi-cal consequences. On the contrary, themajority of researchers believe that thelong history of plant modification by clas-sic breeding provides no evidence of eco-logical disasters (Kareiva 1993). For ex-ample, the most effective gene for TMVresistance in tomato (T/n-i?2) derived frominterspecific crosses between L peruvian-um (donor) and L esculentum (receiver)(Alexander and Cirulli 1966). Althoughthis gene has been extensively incorporat-ed into hundreds of tomato cultivars ofworldwide distribution, there is no evi-dence that it has moved into any of thewild Solanaceae. It is obvious that withnatural or "man-assembled" genes, thepossibility of interspecific or intergenericgene-transfer is the same (Prowidenti andGonsalves 1993).ReferencesAlexander LJ and Cirulli M, 1966. Inheritance of resis-tance to tobacco mosaic virus in tomato. Phytopathol-ogy 56:869.Beachy RN, Loesh-Fries S, and Turner NE, 1990. Coatprotein-mediated resistance against virus infection.Annu Rev Phytopathol 28:451-474.Gonsalves D, Prowidenti R, and Edwards MC, 1982. To-mato white leaf: the relation of an apparent satelliteRNA and cucumber mosaic virus. Phytopathology 72:1533-1538.Gonsalves C, Xue B, Yep M, Fuchs M, Ling K, Namba S,Chee P, Slightom JL, and Gonsalves D, 1994. Transfer-ring cucumber mosaic virus-white leaf strain coat pro-tein gene into Cucumis melo L, and evaluating trans-genic plants for protection against infections. J Am SocHortSci 119:345-355.Grumet R, 1990. Genetically engineered plant virus re-sistance. HortScience 25:508-513.Kareiva P, 1993. Transgenic plants on trial. Nature 363:580-581.Kariyama T, Kuniyasu K, and Mochizuki H, 1971. Stud-ies on the breeding of disease resistance in tomatoesby interspecific hybridization. II. Fertility and diseaseresistance in the progenies of interspecific hybridiza-tion. Bull Hort Res Sta B (Okitsu) 11:33-60.Kearney CM, Gonsalves D, and Prowidenti R, 1990. Asevere strain of cucumber mosaic virus from China andits associated satellite RNA. Plant Dis 74:819-823.Latterot H, 1980. Recherches sur la tomate. In: Report1979-80 de la Station d'Amelioration des Plantes Mar-aicheres d'Avignon. Montfavet, France; 110-112.Leemans J, 1993. Ti to tomato, tomato to market. Bio/Technology 11:S22-S26.Namba S, Ling K, Gonsalves C, Gonsalves D, and Sligh-tom JL, 1991. Expression of the gene encoding the coatprotein of cucumber mosaic virus (CMV) strain WL ap-pears to provide protection to tobacco plants againstinfection by several different CMV strains. Gene 107:181-188.Palukaitis P, 1988. Pathogenicity regulation by satelliteProwidenti and Gonsalves • Resistance to Cucumber Mosaic Virus 87RNAs of cucumber mosaic virus: minor nucleotide se- gress of the Society for Advancement of Breeding Re- sistance to pepper and tomato viruses. In: Resistancequence changes alter host responses. Mol Plant Microb search in Asia and Oceania (SABRAO). Taipei, Taiwan; to viral diseases of vegetables, genetics and breedingInteract 1:75—181. 18. (Kyle MM, ed). Portland: Timber Press; 80-101.Phills BRR, Prowidenti R, and Robinson RW, 1977. Re- Quemada HD, Gonsalves D, and Slightom JL, 1991. Ex- Xue B, Gonsalves C, Prowidenti R, Slightom JL, andaction of Solanum lycopersicoides to viral diseases of pression of a coat protein gene from cucumber mosaic Gonsalves D, 1994. A transgenic tomato expressing atomato. Tomato Genet Coop 27:18. virus strain C in tobacco: protection against infection high level of resistance to strains and serotypes of cu-Prowidenti R and Gonsalves D, 1993. Recent advances !* CfMV ,s'rfns " ^ mechanically or by aphids. cumber mosaic virus. Plant Dis 78:1038-1041.in biotechnological research in relation to plant breed- Phytopathology 8 . /94-8UV. Received March 25, 1994ing. In: The Proceedings of the 7th International Con- Watterson JC, 1993. Development and breeding of re- Accepted September 28, 199488 The Journal of Heredity 1995:86(2)

Inheritance of Resistance to cucumber Mosaic Virus in a Transgenic Tomato Line Expressing the Coat Protein Gene of the White Leaf Strain

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